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Biomol GmbH leupeptin
Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor <t>(leupeptin),</t> 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p
Leupeptin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Post-Translational Control of IL-1? via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53"

Article Title: Post-Translational Control of IL-1? via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003536

Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor (leupeptin), 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p
Figure Legend Snippet: Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor (leupeptin), 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p

Techniques Used: Confocal Microscopy, Immunostaining, Transduction, Expressing, Blocking Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activity Assay, Fluorescence, Incubation, Staining, Protease Inhibitor, High Throughput Screening Assay, Microscopy, Imaging

2) Product Images from "Palmitoylation Controls Recycling in Lysosomal Sorting and Trafficking"

Article Title: Palmitoylation Controls Recycling in Lysosomal Sorting and Trafficking

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/j.1600-0854.2008.00814.x

Palmitoylation is required for the interaction of the lysosomal sorting receptors with retromer. A) HeLa cells were transfected with sortilin-myc, treated with 2-FPA and immunoprecipitated with sortilin-myc or CI-MPR and blotted for the proteins indicated. B) HeLa cells were transfected with sortilin-myc or sortilinC783S-myc and immunoprecipitated with myc antibodies and blotted for the proteins indicated. C) HeLa cells were transfected with sortilin-myc and treated with the palmitoylation inhibitor 2-FPA. The cells were subsequently treated with 50 μg/mL of cycloheximide for the times indicated. D) Wild-type sortilin-myc and sortilinC783S-myc were transfected into HeLa cells and a cycloheximide treatment was performed as in (A). E) A cycloheximide treatment as in (C) was performed in the presence of the lysosomal inhibitors leupeptin and E64. F) A cycloheximide treatment was performed as in (D) in the presence of the lysosomal inhibitors leupeptin and E64. Actin served as a loading control in panels (C), (D), (E) and (F). Ab, antibody; IP, immunoprecipitation; Wt, wild type; Wb, western blotting.
Figure Legend Snippet: Palmitoylation is required for the interaction of the lysosomal sorting receptors with retromer. A) HeLa cells were transfected with sortilin-myc, treated with 2-FPA and immunoprecipitated with sortilin-myc or CI-MPR and blotted for the proteins indicated. B) HeLa cells were transfected with sortilin-myc or sortilinC783S-myc and immunoprecipitated with myc antibodies and blotted for the proteins indicated. C) HeLa cells were transfected with sortilin-myc and treated with the palmitoylation inhibitor 2-FPA. The cells were subsequently treated with 50 μg/mL of cycloheximide for the times indicated. D) Wild-type sortilin-myc and sortilinC783S-myc were transfected into HeLa cells and a cycloheximide treatment was performed as in (A). E) A cycloheximide treatment as in (C) was performed in the presence of the lysosomal inhibitors leupeptin and E64. F) A cycloheximide treatment was performed as in (D) in the presence of the lysosomal inhibitors leupeptin and E64. Actin served as a loading control in panels (C), (D), (E) and (F). Ab, antibody; IP, immunoprecipitation; Wt, wild type; Wb, western blotting.

Techniques Used: Transfection, Immunoprecipitation, Western Blot

3) Product Images from "Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein"

Article Title: Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein

Journal: Journal of Cell Science

doi: 10.1242/jcs.055160

Pulse-chase analysis of wt- and mutant-PLP degradation. (A) HeLa cells were left uninduced (–) or induced to express wt-, W162L-, A242V- or G245A-PLPmh. Cells were treated for 1 hour with DMSO, Z-LLF-CHO (10 μM) or a mixture of leupeptin
Figure Legend Snippet: Pulse-chase analysis of wt- and mutant-PLP degradation. (A) HeLa cells were left uninduced (–) or induced to express wt-, W162L-, A242V- or G245A-PLPmh. Cells were treated for 1 hour with DMSO, Z-LLF-CHO (10 μM) or a mixture of leupeptin

Techniques Used: Pulse Chase, Mutagenesis, Plasmid Purification

4) Product Images from "Proteasome Inhibitor PS-341 (Bortezomib) Induces Calpain-dependent I?B? Degradation *"

Article Title: Proteasome Inhibitor PS-341 (Bortezomib) Induces Calpain-dependent I?B? Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.072694

PS-341 induces caspase-independent ( A ), calpain-mediated ( B–D ) IκBα proteolysis. A , top panel , the indicated cell lines were treated with 50 n m PS-341 alone or 50 n m PS-341 plus 50 μ m Z-VAD-FMK, and 50 μ m leupeptin,
Figure Legend Snippet: PS-341 induces caspase-independent ( A ), calpain-mediated ( B–D ) IκBα proteolysis. A , top panel , the indicated cell lines were treated with 50 n m PS-341 alone or 50 n m PS-341 plus 50 μ m Z-VAD-FMK, and 50 μ m leupeptin,

Techniques Used:

5) Product Images from "Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide"

Article Title: Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide

Journal: Investigational New Drugs

doi: 10.1007/s10637-018-0569-x

a Potent inhibition of proteasome activity by the hexameric 4A6 peptide. Luminescent cell-based proteasome assay measuring inhibition of chymotrypsin-like proteasome activity in intact THP1 cells after 1 h exposure to BTZ, the hexameric peptide 4A6, 4A6-dimer, the hexameric peptide 4E11 and, as control, the tripeptide P121/Reversin, a peptide-based transport inhibitor of the MDR protein P-glycoprotein. Results represent the mean of 3 experiments ± S.D. b Inhibition of chymotrypsin-like but not caspase-like and trypsin-like proteasomal activity by 4A6 . Chymotrypsin-like, caspase-like and trypsin-like proteasomal activities were determined with specific fluorogenic peptide substrates in cell extracts of THP1 cells after 1 h exposure to the indicated concentrations of 4A6. Controls for selective inhibition of chymotrypsin-like, caspase-like and trypsin-like activity included BTZ (10 nM), Ac-APnLP (25 μM) and leupeptin (20 μM), respectively. Results represent the mean of 3 separate experiments ± S.D
Figure Legend Snippet: a Potent inhibition of proteasome activity by the hexameric 4A6 peptide. Luminescent cell-based proteasome assay measuring inhibition of chymotrypsin-like proteasome activity in intact THP1 cells after 1 h exposure to BTZ, the hexameric peptide 4A6, 4A6-dimer, the hexameric peptide 4E11 and, as control, the tripeptide P121/Reversin, a peptide-based transport inhibitor of the MDR protein P-glycoprotein. Results represent the mean of 3 experiments ± S.D. b Inhibition of chymotrypsin-like but not caspase-like and trypsin-like proteasomal activity by 4A6 . Chymotrypsin-like, caspase-like and trypsin-like proteasomal activities were determined with specific fluorogenic peptide substrates in cell extracts of THP1 cells after 1 h exposure to the indicated concentrations of 4A6. Controls for selective inhibition of chymotrypsin-like, caspase-like and trypsin-like activity included BTZ (10 nM), Ac-APnLP (25 μM) and leupeptin (20 μM), respectively. Results represent the mean of 3 separate experiments ± S.D

Techniques Used: Inhibition, Activity Assay

6) Product Images from "Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide"

Article Title: Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide

Journal: Investigational New Drugs

doi: 10.1007/s10637-018-0569-x

a Potent inhibition of proteasome activity by the hexameric 4A6 peptide. Luminescent cell-based proteasome assay measuring inhibition of chymotrypsin-like proteasome activity in intact THP1 cells after 1 h exposure to BTZ, the hexameric peptide 4A6, 4A6-dimer, the hexameric peptide 4E11 and, as control, the tripeptide P121/Reversin, a peptide-based transport inhibitor of the MDR protein P-glycoprotein. Results represent the mean of 3 experiments ± S.D. b Inhibition of chymotrypsin-like but not caspase-like and trypsin-like proteasomal activity by 4A6 . Chymotrypsin-like, caspase-like and trypsin-like proteasomal activities were determined with specific fluorogenic peptide substrates in cell extracts of THP1 cells after 1 h exposure to the indicated concentrations of 4A6. Controls for selective inhibition of chymotrypsin-like, caspase-like and trypsin-like activity included BTZ (10 nM), Ac-APnLP (25 μM) and leupeptin (20 μM), respectively. Results represent the mean of 3 separate experiments ± S.D
Figure Legend Snippet: a Potent inhibition of proteasome activity by the hexameric 4A6 peptide. Luminescent cell-based proteasome assay measuring inhibition of chymotrypsin-like proteasome activity in intact THP1 cells after 1 h exposure to BTZ, the hexameric peptide 4A6, 4A6-dimer, the hexameric peptide 4E11 and, as control, the tripeptide P121/Reversin, a peptide-based transport inhibitor of the MDR protein P-glycoprotein. Results represent the mean of 3 experiments ± S.D. b Inhibition of chymotrypsin-like but not caspase-like and trypsin-like proteasomal activity by 4A6 . Chymotrypsin-like, caspase-like and trypsin-like proteasomal activities were determined with specific fluorogenic peptide substrates in cell extracts of THP1 cells after 1 h exposure to the indicated concentrations of 4A6. Controls for selective inhibition of chymotrypsin-like, caspase-like and trypsin-like activity included BTZ (10 nM), Ac-APnLP (25 μM) and leupeptin (20 μM), respectively. Results represent the mean of 3 separate experiments ± S.D

Techniques Used: Inhibition, Activity Assay

Related Articles

Protease Inhibitor:

Article Title: Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein
Article Snippet: .. Media and reagents were from Lonza (Wokingham, UK), Z-LLF-CHO from Calbiochem, protease-inhibitor cocktail from Sigma, and leupeptin, pepstatin A and lactacystin from BIOMOL (Exeter, UK). ..

Incubation:

Article Title: Post-Translational Control of IL-1? via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53
Article Snippet: .. Inhibitors of autophagy used: 1 mM 3-methyladenine (Enzo Life Sciences) incubated for 8 h and 100 nM bafilomycin (Enzo Life Sciences) incubated for 8 h. Inhibitors of lysosome/protease activity: 20 µM of Calpain inhibitor PD 150,606 (Adipogen), 25 µM of cathepsin B inhibitor CA-074 (Enzo Life Sciences), 10 µM of Leupeptin (Biomol), 20 µM Vincristine (Enzo Life Sciences). .. All treatments were carried out for 6 h. Starvation was done by cultivating the cells in HEPES buffered saline solution (HBSS) for 8 h. For proteasomal inhibition cells were treated with 5 µM of the proteasome inhibitor MG132 (Calbiochem) for 6 h. Quantification of autophagy, lysosome amount and activity as well as cathepsin B activity were performed using high throughput high resolution fluorescence microscopy analysis (BD pathway, Beckton Dickenson) using the filter set Ex: 516 nm/Em: 590 nm.

Activity Assay:

Article Title: Post-Translational Control of IL-1? via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53
Article Snippet: .. Inhibitors of autophagy used: 1 mM 3-methyladenine (Enzo Life Sciences) incubated for 8 h and 100 nM bafilomycin (Enzo Life Sciences) incubated for 8 h. Inhibitors of lysosome/protease activity: 20 µM of Calpain inhibitor PD 150,606 (Adipogen), 25 µM of cathepsin B inhibitor CA-074 (Enzo Life Sciences), 10 µM of Leupeptin (Biomol), 20 µM Vincristine (Enzo Life Sciences). .. All treatments were carried out for 6 h. Starvation was done by cultivating the cells in HEPES buffered saline solution (HBSS) for 8 h. For proteasomal inhibition cells were treated with 5 µM of the proteasome inhibitor MG132 (Calbiochem) for 6 h. Quantification of autophagy, lysosome amount and activity as well as cathepsin B activity were performed using high throughput high resolution fluorescence microscopy analysis (BD pathway, Beckton Dickenson) using the filter set Ex: 516 nm/Em: 590 nm.

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    Biomol GmbH leupeptin
    Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor <t>(leupeptin),</t> 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p
    Leupeptin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leupeptin/product/Biomol GmbH
    Average 92 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    leupeptin - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    90
    Biomol GmbH leupeptine
    Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the white muscle extract . Figure 8b shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg white muscle-extract and 25 μg ubiquitylprotein-isopeptidase (see material). The batches incubated with ubiquityl-isopeptidase had a final concentration of 50 mM tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml <t>leupeptine,</t> pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin after ubiquitylprotein-isopeptidase incubation.
    Leupeptine, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leupeptine/product/Biomol GmbH
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    leupeptine - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

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    Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor (leupeptin), 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p

    Journal: PLoS Pathogens

    Article Title: Post-Translational Control of IL-1? via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53

    doi: 10.1371/journal.ppat.1003536

    Figure Lengend Snippet: Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation. A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor (leupeptin), 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p

    Article Snippet: Inhibitors of autophagy used: 1 mM 3-methyladenine (Enzo Life Sciences) incubated for 8 h and 100 nM bafilomycin (Enzo Life Sciences) incubated for 8 h. Inhibitors of lysosome/protease activity: 20 µM of Calpain inhibitor PD 150,606 (Adipogen), 25 µM of cathepsin B inhibitor CA-074 (Enzo Life Sciences), 10 µM of Leupeptin (Biomol), 20 µM Vincristine (Enzo Life Sciences).

    Techniques: Confocal Microscopy, Immunostaining, Transduction, Expressing, Blocking Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activity Assay, Fluorescence, Incubation, Staining, Protease Inhibitor, High Throughput Screening Assay, Microscopy, Imaging

    Palmitoylation is required for the interaction of the lysosomal sorting receptors with retromer. A) HeLa cells were transfected with sortilin-myc, treated with 2-FPA and immunoprecipitated with sortilin-myc or CI-MPR and blotted for the proteins indicated. B) HeLa cells were transfected with sortilin-myc or sortilinC783S-myc and immunoprecipitated with myc antibodies and blotted for the proteins indicated. C) HeLa cells were transfected with sortilin-myc and treated with the palmitoylation inhibitor 2-FPA. The cells were subsequently treated with 50 μg/mL of cycloheximide for the times indicated. D) Wild-type sortilin-myc and sortilinC783S-myc were transfected into HeLa cells and a cycloheximide treatment was performed as in (A). E) A cycloheximide treatment as in (C) was performed in the presence of the lysosomal inhibitors leupeptin and E64. F) A cycloheximide treatment was performed as in (D) in the presence of the lysosomal inhibitors leupeptin and E64. Actin served as a loading control in panels (C), (D), (E) and (F). Ab, antibody; IP, immunoprecipitation; Wt, wild type; Wb, western blotting.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Palmitoylation Controls Recycling in Lysosomal Sorting and Trafficking

    doi: 10.1111/j.1600-0854.2008.00814.x

    Figure Lengend Snippet: Palmitoylation is required for the interaction of the lysosomal sorting receptors with retromer. A) HeLa cells were transfected with sortilin-myc, treated with 2-FPA and immunoprecipitated with sortilin-myc or CI-MPR and blotted for the proteins indicated. B) HeLa cells were transfected with sortilin-myc or sortilinC783S-myc and immunoprecipitated with myc antibodies and blotted for the proteins indicated. C) HeLa cells were transfected with sortilin-myc and treated with the palmitoylation inhibitor 2-FPA. The cells were subsequently treated with 50 μg/mL of cycloheximide for the times indicated. D) Wild-type sortilin-myc and sortilinC783S-myc were transfected into HeLa cells and a cycloheximide treatment was performed as in (A). E) A cycloheximide treatment as in (C) was performed in the presence of the lysosomal inhibitors leupeptin and E64. F) A cycloheximide treatment was performed as in (D) in the presence of the lysosomal inhibitors leupeptin and E64. Actin served as a loading control in panels (C), (D), (E) and (F). Ab, antibody; IP, immunoprecipitation; Wt, wild type; Wb, western blotting.

    Article Snippet: The 2-FPA, leupeptin and E64 were purchased from Biomol.

    Techniques: Transfection, Immunoprecipitation, Western Blot

    Pulse-chase analysis of wt- and mutant-PLP degradation. (A) HeLa cells were left uninduced (–) or induced to express wt-, W162L-, A242V- or G245A-PLPmh. Cells were treated for 1 hour with DMSO, Z-LLF-CHO (10 μM) or a mixture of leupeptin

    Journal: Journal of Cell Science

    Article Title: Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein

    doi: 10.1242/jcs.055160

    Figure Lengend Snippet: Pulse-chase analysis of wt- and mutant-PLP degradation. (A) HeLa cells were left uninduced (–) or induced to express wt-, W162L-, A242V- or G245A-PLPmh. Cells were treated for 1 hour with DMSO, Z-LLF-CHO (10 μM) or a mixture of leupeptin

    Article Snippet: Media and reagents were from Lonza (Wokingham, UK), Z-LLF-CHO from Calbiochem, protease-inhibitor cocktail from Sigma, and leupeptin, pepstatin A and lactacystin from BIOMOL (Exeter, UK).

    Techniques: Pulse Chase, Mutagenesis, Plasmid Purification

    Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the white muscle extract . Figure 8b shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg white muscle-extract and 25 μg ubiquitylprotein-isopeptidase (see material). The batches incubated with ubiquityl-isopeptidase had a final concentration of 50 mM tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin after ubiquitylprotein-isopeptidase incubation.

    Journal: European Journal of Medical Research

    Article Title: Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    doi: 10.1186/2047-783X-15-10-428

    Figure Lengend Snippet: Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the white muscle extract . Figure 8b shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg white muscle-extract and 25 μg ubiquitylprotein-isopeptidase (see material). The batches incubated with ubiquityl-isopeptidase had a final concentration of 50 mM tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin after ubiquitylprotein-isopeptidase incubation.

    Article Snippet: Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg).

    Techniques: Incubation, Concentration Assay

    Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the heart muscle extract Figure 7a shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg heart muscle-extract and 25 μg ubiquitylprotein-isopeptidase (see material) . The batches incubated with ubiquitylprotein-isopeptidases had a final concentration of 50 mM Tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. Both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin.

    Journal: European Journal of Medical Research

    Article Title: Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    doi: 10.1186/2047-783X-15-10-428

    Figure Lengend Snippet: Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the heart muscle extract Figure 7a shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg heart muscle-extract and 25 μg ubiquitylprotein-isopeptidase (see material) . The batches incubated with ubiquitylprotein-isopeptidases had a final concentration of 50 mM Tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. Both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin.

    Article Snippet: Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg).

    Techniques: Incubation, Concentration Assay

    Trypsin-incubation of white muscle extract and APFII . Figure 10a shows the cleavage of internal and external ubiquitin-conjugates of the white muscle with trypsin. The incubation batches have a final concentration of 5 μg/ml leupeptine. The batches were incubated for 30 min in 37°C in a waterbath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet were solved in the sample buffer for Laemmli sytem and then blotted on PVDF-membrane (see materials). Lane 1: 8.5 μg ubiquityl-calmodulin conjugates and 2 μg unconjugated ubiquitin. Lane 2: 200 μg white muscle extract, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 3: 200 μg white muscle extract and 65 μg trypsin. Lane 4: 200 μg white muscle extract. Lane 5: 200 μg white muscle APFII, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 6: 200 μg white muscle APFII and 65 μg trypsin. Lane 7: 200 μg white muscle APFII.

    Journal: European Journal of Medical Research

    Article Title: Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    doi: 10.1186/2047-783X-15-10-428

    Figure Lengend Snippet: Trypsin-incubation of white muscle extract and APFII . Figure 10a shows the cleavage of internal and external ubiquitin-conjugates of the white muscle with trypsin. The incubation batches have a final concentration of 5 μg/ml leupeptine. The batches were incubated for 30 min in 37°C in a waterbath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet were solved in the sample buffer for Laemmli sytem and then blotted on PVDF-membrane (see materials). Lane 1: 8.5 μg ubiquityl-calmodulin conjugates and 2 μg unconjugated ubiquitin. Lane 2: 200 μg white muscle extract, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 3: 200 μg white muscle extract and 65 μg trypsin. Lane 4: 200 μg white muscle extract. Lane 5: 200 μg white muscle APFII, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 6: 200 μg white muscle APFII and 65 μg trypsin. Lane 7: 200 μg white muscle APFII.

    Article Snippet: Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg).

    Techniques: Incubation, Concentration Assay, Centrifugation

    Trypsin-incubation of heart muscle-extract and APFII . Figure 9a shows the cleavage of internal and external ubiquitin-conjugates of the heart muscle with trypsin. The incubation batches have a final concentration of 5 μg/ml leupeptine. The batches were incubated for 30 min in 37°C in a waterbath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet were solved in the sample buffer for Laemmli system and then blotted on PVDF-membrane (see materials). Lane 1: 8.5 μg ubiquityl-calmodulin conjugates and 2 μg unconjugated ubiquitin. Lane 2: 200 μg red muscle-extract, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 3: 200 μg heart muscle extract and 65 μg trypsin. Lane 4: 200 μg heart muscle extract. Lane 5: 200 μg heart muscle APFII, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 6: 200 μg heart muscle APFII and 65 μg trypsin. Lane 7: 200 μg heart muscle APFII.

    Journal: European Journal of Medical Research

    Article Title: Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    doi: 10.1186/2047-783X-15-10-428

    Figure Lengend Snippet: Trypsin-incubation of heart muscle-extract and APFII . Figure 9a shows the cleavage of internal and external ubiquitin-conjugates of the heart muscle with trypsin. The incubation batches have a final concentration of 5 μg/ml leupeptine. The batches were incubated for 30 min in 37°C in a waterbath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet were solved in the sample buffer for Laemmli system and then blotted on PVDF-membrane (see materials). Lane 1: 8.5 μg ubiquityl-calmodulin conjugates and 2 μg unconjugated ubiquitin. Lane 2: 200 μg red muscle-extract, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 3: 200 μg heart muscle extract and 65 μg trypsin. Lane 4: 200 μg heart muscle extract. Lane 5: 200 μg heart muscle APFII, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 6: 200 μg heart muscle APFII and 65 μg trypsin. Lane 7: 200 μg heart muscle APFII.

    Article Snippet: Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg).

    Techniques: Incubation, Concentration Assay, Centrifugation

    Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the white muscle APFII Figure 8c shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg white muscle-APFII and 25 μg ubiquitylprotein-isopeptidase (see material) . The batches incubated with ubiquityl-isopeptidase had a final concentration of 50 mM tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°c). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin.

    Journal: European Journal of Medical Research

    Article Title: Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    doi: 10.1186/2047-783X-15-10-428

    Figure Lengend Snippet: Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the white muscle APFII Figure 8c shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg white muscle-APFII and 25 μg ubiquitylprotein-isopeptidase (see material) . The batches incubated with ubiquityl-isopeptidase had a final concentration of 50 mM tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°c). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin.

    Article Snippet: Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg).

    Techniques: Incubation, Concentration Assay

    Cleavage of Ubiquitin-conjugates in the white muscle with ubiquitylprotein-Isopeptidase . Figure 8a shows the cleavage of ubiquitin-conjugates of the extract and APFII after anion-exchange chromatography from the white muscle (batch 2-7). The incubation batches contained with a final concentration of 5 μg/ml leupeptine and depending on batch 5 mM iodacetamide. All batches were incubated for 60 min in 37°C in a waterbath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet was solved in sample buffer for Laemmli system and was blotted on PVDF-membrane (see Materials). Lane 1: 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) (control). Lane 2: 200 μg white muscle extract, 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) and 25 μg ubiquitylprotein-isopeptidase Lane 3: 200 μg white muscle extract and 25 μg ubiquitylprotein-isopeptidase. Lane 4: 200 μg white muscle extract and with a final concentration of 5 mM iodacetamide. Lane 5: 200 μg white muscle run-through, 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) and 25 μg ubiquitylprotein-isopeptidase. Lane 6 200 μg white muscle run-through a nd 25 μg ubiquitylprotein-isopeptidase. Lane 7: 200 μg white muscle run-through (control) and 5 mM iodacetamide.

    Journal: European Journal of Medical Research

    Article Title: Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    doi: 10.1186/2047-783X-15-10-428

    Figure Lengend Snippet: Cleavage of Ubiquitin-conjugates in the white muscle with ubiquitylprotein-Isopeptidase . Figure 8a shows the cleavage of ubiquitin-conjugates of the extract and APFII after anion-exchange chromatography from the white muscle (batch 2-7). The incubation batches contained with a final concentration of 5 μg/ml leupeptine and depending on batch 5 mM iodacetamide. All batches were incubated for 60 min in 37°C in a waterbath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet was solved in sample buffer for Laemmli system and was blotted on PVDF-membrane (see Materials). Lane 1: 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) (control). Lane 2: 200 μg white muscle extract, 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) and 25 μg ubiquitylprotein-isopeptidase Lane 3: 200 μg white muscle extract and 25 μg ubiquitylprotein-isopeptidase. Lane 4: 200 μg white muscle extract and with a final concentration of 5 mM iodacetamide. Lane 5: 200 μg white muscle run-through, 8.5 μg ubiquitin-calmodulin conjugates (Order 1-3) and 25 μg ubiquitylprotein-isopeptidase. Lane 6 200 μg white muscle run-through a nd 25 μg ubiquitylprotein-isopeptidase. Lane 7: 200 μg white muscle run-through (control) and 5 mM iodacetamide.

    Article Snippet: Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg).

    Techniques: Chromatography, Incubation, Concentration Assay, Centrifugation

    Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the heart muscle APFII Figure 7b shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg heart muscle-APFII and 25 μg ubiquitylprotein-isopeptidase (see material) . The batches incubated with ubiquitylprotein-isopeptidase had a final concentration of 50 mM Tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM M iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. Both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin.

    Journal: European Journal of Medical Research

    Article Title: Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    doi: 10.1186/2047-783X-15-10-428

    Figure Lengend Snippet: Overlay of the densitogram curves before and after ubiquitylprotein-isopeptidase incubation of the heart muscle APFII Figure 7b shows the densitogram curves before (green lane) and after (red lane) enzyme incubation of 200 μg heart muscle-APFII and 25 μg ubiquitylprotein-isopeptidase (see material) . The batches incubated with ubiquitylprotein-isopeptidase had a final concentration of 50 mM Tris/HCl, 1 mM DTE, 50 μM PMSF and 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min) in a waterbath the reaction was irreversible inhibited by an final concentration of 5 mM M iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase. Both densitograms were overlayed to estimate the release of unconjugated (free) ubiquitin.

    Article Snippet: Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg).

    Techniques: Incubation, Concentration Assay